(1) Field of the Invention
The present invention relates to treatments for osteoporosis and other disorders characterized by bone loss or insufficient bone mass. More specifically, the present invention relates to the cynomolgus monkey Dickkopf-4 (cDkk-4) protein, to isolated nucleic acid molecules which encode the cDkk-4 protein, and to recombinant vectors and hosts comprising DNA encoding the cDkk-4 protein. The present invention further relates to methods for identifying analytes which interfere with the interaction between the cDkk-4 protein and a homologous or heterologous Dkk receptor and analytes which interfere with the interaction and stimulate bone formation. Analytes identified using one or more of the methods are useful for treating osteoporosis and other bone loss disorders.
(2) Description of Related Art
The skeletal disorder osteoporosis is the leading cause of morbidity in the elderly. Osteoporosis is characterized by bone loss resulting from an imbalance between bone resorption (destruction) and bone formation. This condition leads to an increased risk of bone fractures, which may occur following low levels of trauma. In the United States, there are currently about 20 million people with detectable fractures of the vertebrae due to osteoporosis. Mortality due to bone fractures is not uncommon among the elderly patient population
Elderly, post-menopausal women are at the highest risk of developing osteoporosis due to a deficiency of estrogen, which is necessary for proper bone maintenance. Insufficient estrogen levels lead to increased production and longevity of destructive osteoclasts, which in turn, leads to increased bone resorption. As a result, an average of 5% bone loss is observed in the vertebrae per year. Although less common, osteoporosis also affects elderly men. The existence of osteoporosis in elderly men may also be due, in part, to insufficient estrogen levels caused by a decrease in circulating testosterone.
Therapeutic strategies for overcoming bone loss include both the prevention of bone resorption and the stimulation of bone growth. The majority of therapeutic targets that have led to efficacious osteoporosis treatments fall into the former category. Thus, the first line of treatment/prevention of this condition has historically been the inhibition of bone resorption using compounds such as bisphosphonates, estrogens, selective estrogen receptor modulators (SERMs), and calcitonin. Because inhibition of bone resorption cannot restore bone mass, this approach is an ineffective treatment for patients who have already lost a significant amount of bone (>30%). Additionally, the effectiveness of osteoporosis treatments that function by this mechanism is not consistent across the skeletal anatomy because the rate of bone turnover differs from one site to another. For example, the bone turnover rate is higher in the trabecular bone of the vertebrae than in the cortex of the long bones; thus, bone resorption inhibitors are less effective in increasing hip bone mineral density (BMD) and preventing hip fracture. Therefore, osteoanabolic agents, which increase cortical/periosteal bone formation and bone mass at long bones, would address an unmet need in the treatment of osteoporosis, especially for patients with high risk of hip fractures.
One potential therapeutic target for metabolic disorders, including osteoporosis, is the low-density lipoprotein receptor related protein 5 (LRP5). LRP5 belongs to the low density lipoprotein receptor (LDLR) gene family of cell surface receptors, characterized by cysteine-rich, complement-type LDLR ligand binding domains. LRP5 was isolated based on its proximity to the locus of osteoporosis pseudoglioma syndrome (OPG), an autosomal recessive disorder characterized by severe osteoporosis (Hey et al., Gene 216: 103-111 (1998); Todd et al., WO 98/46743). Additional support for the notion that LRP5 represents a therapeutic target for osteoporosis comes from the observation that loss-of-function mutations of LRP5 lead to OPG (Gong et al., Cell 107: 513-523 (2001)).
Interestingly, aberrant expression of LRP5 is also associated with high bone mass trait (HBM), an autosomal dominant human genetic skeletal condition characterized by strilingly increased bone mass. Positional cloning of the HBM mutation demonstrated that HBM results from a G171V mutation of the LRP5 gene that leads to a gain of function (Little et al., Am. J. Hum. Genet. 70: 11-19 (2002); Boyden et al., N. Eng. J. Med. 346: 1513-1521 (2002)). These complementary findings, together with the finding that null mutations of LRP5 in mice results in severe bone loss (Kato, J. Cell Biol. 157: 303-314 (2002)), suggest that LRP5 has an important role in bone formation and bone mass in humans.
Despite its specific role in stimulating bone growth, the LRP5 gene was shown to have a nearly ubiquitous expression profile. The mechanism by which activation of LRP5 leads to osteogenesis is not known. However, at the molecular level, it was recently shown that LRP5 and closely related LRP6 are involved in Wingless (Wnt) signaling as co-receptors for Wnt. Wnt activated genes encode secreted proteins implicated in a diverse array of developmental and adult physiological processes, such as mediating cell growth and differentiation in the central nervous system. It was also shown that LRP5 and LRP6 are receptors for the secreted protein Dickkopf-1 (Dkk-1) and that their association with Dkk-1 represses Wnt signaling (Mao et al., Nature 411: 321-325 (2001); Semenov et al., Curr. Biol. 11: 951-961 (2001); Bafico et al., Nat. Cell Biol. 3: 683-686 (2001)).
Dickkopf-1 is a secreted protein that participates in embryonic head induction and antagonizes Wnt signaling (Glinka et al., Nature 391: 357-362 (1998)). The amino acid sequence of human Dkk-1 protein and the nucleotides encoding it have been described in WO 00/52047 to McCarthy and Krupnick et al., Gene 238: 301-313(1999). Expression of Dkk-1 in humans was thought to be restricted to the placenta, suggesting a role for Dkk-1 in embryonic development (Krupnick et al., supra). WO 02/092015 to Allen et al. describes assays relating to the interaction between LRP5, HBM (a variant of LRP5), or LRP6 with Dkk-1. In humans, Dickkopf-1 is a member of a Dickkopf gene family which at present has been shown to include Dkk-1, Dkk-2, Dkk-3, and Dkk-4 (Krupnick et al., supra). While Dkk-1 and Dkk-4, but not Dkk-2 or Dkk-3, have been shown to suppress Wnt-induced secondary axis induction in Xenopus embryos, neither block axis induction triggered either by Xenopus Dishevelled protein or Frizzled protein which suggests that their Wnt inhibitory activity is by a mechanism upstream of the Frizzled protein's position in the Wnt signaling pathway (Krupnick et al., supra). It has been suggested that Dkk-1 or Dkk-2 might have an inhibitory effect on bone formation and that either might be a target for the prevention or treatment of osteoporosis (Patel and Karensky, N. Eng. J. Med. 346: 1572-1573 (2002); Boyden et al., N. Eng. J. Med. 346: 1513-1521 (2002)). A role for Dkk-4 in the Wnt signaling pathway is not clear.
In addition to LRP5 and LRP6, recent studies indicate that the transmembrane proteins kremen1 and kremen2 are high-affinity Dkk-1 receptors which functionally cooperate with Dkk-1 to block Wnt signaling, thereby regulating central nervous system patterning during embryonic development (Mao et al., Nature 417: 664-667 (2002); Davidson et al., Development 129: 5587-96 (2002)). Currently, it is believed that Dkk-1 inhibits LRP5 or LRP6 activated Wnt signaling by cooperating with kremen to form a ternary complex with the LRP5 or LRP6. The ternary complex is rapidly endocytosed which removes the LPR5 or LRP6 from the membrane, thus preventing LPR5 or LPR6 from binding Wnt.
Despite our increased understanding of the regulation of bone formation, osteoporosis has remained a leading health concern because traditional therapies, which act by preventing bone loss, are ineffective treatments for patients who have already suffered significant bone loss. Because patients who have lost significant bone tissue have bones which are brittle and easily broken, it would be particularly desirable to have treatments for osteoporosis (and other disorders characterized by bone-loss) which are osteoanabolic, that is, treatments which stimulate bone growth. Therefore, there is a need for a method for identifying compounds or drugs for treating osteoporosis and other bone mass disorders which stimulate bone growth. Such compounds would be an ideal treatment for osteoporosis as a monotherapy or in combination with inhibitors of bone resorption, such as bisphosphonates, estrogens, SERMs, cathepsin K inhibitors, αVβ3 antagonists, calcitonin, proton pump inhibitors.